Novel treatment for cough

ABSTRACT

The invention discloses the existence of cannabinoid receptors in the airways, which are functionally linked to inhibition of cough. Locally acting cannabinoid agents can be administered to the airways of a subject to ameliorate cough, without causing the psychoactive effects characteristic of systemically administered cannabinoids. In addition, locally or systemically administered cannabinoid inactivation inhibitors can also be used to ameliorate cough. The present invention also defines conditions under which cannabinoid agents can be administered to produce anti-tussive effects devoid of bronchial constriction.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application 60/206,591, filed May 23, 2000. Theaforementioned application is explicitly incorporated herein byreference in its entirety and for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under a grant from theNational Institutes of Health. The Government may have certain rights inthis invention.

TECHNICAL FIELD

This invention relates to pharmaceutical compositions for preventing theinitiation of cough and methods for using the compositions for thetreatment of cough. More particularly, the invention relates to thelocal administration of a therapeutically effective amount of apharmaceutical composition comprising at least one direct or indirectcannabinoid receptor agonist to produce an anti-tussive effect withoutsignificant delivery of the agonist(s) to the systemic circulation.

BACKGROUND

Administration of the main active constituent of the cannabis plant,Δ⁹-tetrahydrocannabinol (Δ⁹-THC), produces in animals and humansalleviation of cough and bronchospasm, which suggest a possibleapplication of cannabis-like (cannabinoid) compounds for the treatmentof cough. The potential therapeutic value of this observation ishindered, however, by two factors. First, the systemic administration ofcannabinoid compounds produces significant psychoactive effects (forexample, memory impairment, dysphoria, alteration in the perception oftime, and habit formation). Second, some asthmatic patients who receiveΔ⁹-THC respond to this compound with a paradoxical bronchialconstriction. Therefore, there exists a need for compounds, orpharmaceutical preparations thereof, which can prevent or alleviatecough in animals and humans without producing significant psychoactiveeffects.

SUMMARY OF THE INVENTION

In one general aspect, the present invention discloses the existence ofcannabinoid receptors in the airways, which are functionally linked toinhibition of cough. Further, the invention teaches that localadministration of locally acting cannabinoid agents in the airwaysreduce cough, without causing the psychoactive effects characteristic ofsystemically administered cannabinoids. Even further, the presentinvention defines conditions under which cannabinoid agents can beadministered to produce anti-tussive effects devoid of bronchialconstriction. Specifically, the invention demonstrates that cannabinoidcompounds produce bronchial constriction when the intrinsic constrictingtone of the vagus nerve is reduced or eliminated.

In another aspect, the present invention also teaches the local orsystemic use of cannabinoid inactivation inhibitors, alone or inconjunction with a cannabinoid receptor agonist, to ameliorate cough.

The pharmaceutical compositions and methods of the present invention arecharacterized by their ability to inhibit cough initiation and/orsignaling from the upper airways to the central nervous system.Specifically, the present invention results in the peripheral inhibitionof cough signaling. Without being bound by any theory, it is believedthat the pharmaceutical compositions of the present inventionshort-circuit the intracellular signaling cascade initiating cough byactivating CB1 cannabinoid receptors found in the upper airways ofmammals. The present invention regulates cough signaling at theperiphery by the activation of local CB1 cannabinoid receptors where itis believed that endogenous cannabinoids participate in filteringemerging cough signals within the upper airways. The present inventionunexpectedly achieves the above superior desired anti-tussive effectswithout the dysphoric side effects and habit-forming propertiescharacteristic of centrally acting cannabimimetic or opiate drugs.

The invention provides a method for ameliorating or preventing cough ina subject, wherein the method comprises administration to the subject ofa cannabinoid receptor agonist having anti-tussive properties withoutany significant psychoactive effects. The subject may be animal orhuman. As an example, one method of treating cough in a mammal maycomprise topically administering into the upper airways (for example, byaerosol) an effective amount of at least one locally acting cannabinoidreceptor agonist in a pharmaceutically acceptable excipient for topicaladministration.

In the methods of the invention, a cannabinoid receptor agonist, or apharmaceutical composition thereof, in accordance to the presentinvention can include cannabinoid receptor agonists having the generalformula I:

-   -   wherein    -   X is N—R1 or O;    -   R is a saturated or unsaturated, chiral or achiral, cyclic or        acyclic, substituted or unsubstituted hydrocarbyl group with 11        to 29 carbon atoms, optionally incorporating up to 6 oxygen or        sulfur atoms;    -   R1, R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

When R2 is OH and X is N—H, they may be combined through the carbonylgroup to form a heterocyclic ring structure, e.g. an oxazolidinone ring.Alternatively, when R2 is OH and X is N—H, they may be combined to forma heterocyclic ring structure, e.g. a morpholine ring.

In particular, the invention contemplates a family of amides with thegeneric structure shown below and active as cannabinoid receptoragonists having formula II:

-   -   wherein    -   R is a saturated or unsaturated, substituted or unsubstituted        hydrocarbyl group with from 15 to 29 carbon atoms, optionally        incorporating up to 3 oxygen or sulfur atoms;    -   R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

When R2 is OH and X is N—H, they may be combined to form a heterocyclicring structure.

The present invention also contemplates a family of esters with thegeneric structure shown below and active as cannabinoid receptoragonists having formula III:

-   -   wherein    -   R is a saturated or unsaturated, substituted or unsubstituted        hydrocarbyl group with from 15 to 29 carbon atoms, optionally        incorporating up to 3 oxygen atoms;    -   R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

The invention further contemplates a family of amides with the genericstructure shown below and active as inhibitors of endogenous cannabinoidinactivation in accordance with the present invention having formula IV:

-   -   wherein    -   R is a polyunsaturated, substituted or unsubstituted hydrocarbyl        group with from 18 to 22 carbon atoms;    -   R2 is selected independently from substituted or unsubstituted        cycloalkyl (C3-6) group and substituted or unsubstituted phenyl        group (e.g., p-hydroxyphenyl, p-hydroxy-o-methyl-phenyl).

The invention also contemplates a family of fatty acid derivatives withthe generic structure shown below and active as inhibitors of endogenouscannabinoid inactivation having formula V:R₁—X—R₂

-   -   wherein    -   R1 is a saturated or polyunsaturated, substituted or        unsubstituted hydrocarbyl group with from 6 to 22 carbon atoms;    -   X is —C═O or SO₂—; and    -   R2 is a halogen or a halogen-substituted methyl group.

In accordance with another aspect of the present invention, there aredisclosed pharmaceutical compositions for treating cough comprising thefollowing examples of direct and indirect acting cannabinoid receptoragonists: arachidonylethanolamide (anandamide),(R)-(+)arachidonyl-1¹-hydroxy-2¹-propylamide, cis-7,10,13,16-docosatetraenoylethanolamide, homo-delta-linoleyethanolamide,N-propyl-arachidonylethanolamide, N-ethyl-arachidonylethanolamide, and2-arachidonylglycerol. There are also disclosed pharmaceuticalcompositions for treating cough comprising the following examples ofcannabinoid inactivation inhibitors:N-(4-hydroxyphenyl)-arachidonylamide, palmitylsulphonylfluoride, andarachidonyltrifluoromethylketone.

The cannabinoid receptor agonist and/or cannabinoid inactivationinhibitors, and pharmaceutically acceptable excipient, may, among otherthings, be formulated as an aerosol, spray, or solution, to be inhaled,to be administered orally, or to be administered parenterally, such asintravenously.

The locally acting cannabinoid receptor agonist and/or cannabinoidinactivation inhibitor, and pharmaceutically acceptable excipient, maybe administered in conjunction with at least one additional therapeuticagent from the group consisting of anti-inflammatory compounds, systemicanti-tussives, and local anesthetics.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits cited herein are expressly incorporated by reference forall purposes.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict chemical structures of cannabinoid compounds andinhibitors of endogenous cannabinoid inactivation.

FIG. 1A shows the general chemical structure of the direct-actingcannabinoid compounds of the present invention.

FIG. 1B shows the general chemical structure of a representativecannabinoid inactivation inhibitor (inhibitor of anandamide transport).

FIG. 1C shows the general chemical structure of another representativecannabinoid inactivation inhibitor (inhibitor of anandamide hydrolysis).

FIGS. 2A-C depict bar graphs summarizing data showing that anandamideinhibits capsaicin-evoked bronchospasm and cough in guinea pigs byactivating peripheral CB1-type cannabinoid receptors. Results areexpressed as mean±s.e.m, with n=3 for each condition. Asterisk indicatesP<0.01.

FIG. 2A is a bar graph showing the constricting effect of capsaicin(Caps, mg per kg, intravenous, i.v.) on guinea pig bronchial smoothmuscle and its antagonism by the vanilloid receptor antagonistcapsazepine (Cpz, 0.2 mg per kg, i.v.).

FIG. 2B is a bar graph showing the dose-dependent inhibitory effects ofanandamide (AEA, mg per kg, i.v.) on capsaicin (30 mg per kg)-evokedbronchospasm in the absence or presence of the CB1 antagonist SR141716A(SR1, 0.5 mg per kg, i.v.) or the CB2 antagonist SR144528 (SR2, 0.3 mgper kg, i.v.).

FIG. 2C is a bar graph summarizing data showing the inhibitory effectsof anandamide on capsaicin-evoked cough in the absence or presence ofSR141716A (0.5 mg per kg, i.v.) or SR144528 (0.3 mg per kg, i.v.).

FIGS. 3A-E depict bar graphs and representative tracings of muscletension showing that anandamide causes bronchoconstriction invagotomized, atropine-treated guinea pigs by activating peripheral CB1cannabinoid receptors. Asterisk indicates P<0.01.

FIG. 3A is a bar graph summarizing the dose-dependent effect ofanandamide (AEA, mg per kg, i.v.) on bronchial smooth muscle, in thepresence or absence of the CB1 antagonist SR141716A (SR1, 0.2 mg per kg,i.v.) (n=6 for each condition).

FIG. 3B is a bar graph summarizing the dose-dependent effects ofanandamide (5-30 mg per animal, intratracheal) in the absence orpresence of SR141716A (SR1, 0.3 mg per kg, i.v.) (n=6 for eachcondition). SR141716A was administered 15 min before anandamide.

FIG. 3C is a representative tracing illustrating the effect ofanandamide (100 μM) on isotonic muscle tension in guinea pig parenchymastrips and its reversal by the CB1 antagonist SR141716A (1 μM).

FIG. 3D is a response of the same lung strip of FIG. 3C to histamine(His, 10 μM) shown for comparison. The upward deflections are caused byrepeated washes of the lung strip.

FIG. 3E is a bar graph summarizing the dose-dependent contractions oflung parenchyma in the presence of anandamide (μM) and antagonism ofthis effect by SR141716A (1 μM) (n=6 for each condition).

FIGS. 4A-G show localization of CB1 cannabinoid receptors on axonterminals and preterminal segments in rat lungs.

FIGS. 4A-B show silver-intensified gold particles labeling CB1cannabinoid receptors, indicated by thin arrows, on serial sections ofaxon terminals in a bronchiole. Axon terminals containing smallelectron-translucent and large dense-core vesicles (thick arrow) areembedded in the collagen matrix and are surrounded by bronchial smoothmuscle cells (BSM).

FIG. 4C shows that, in some cases, cannabinoid receptor labeling wasobserved in proximity of vesicle clusters (arrowhead), indicatingputative neurotransmitter release sites.

FIGS. 4D-E show serial sections in the adventitious layer revealmultiple axon terminals packed together into glial capsules.

FIGS. 4F-G show co-localization of cannabinoid receptor and neuropeptideY (NPY) immunoreactivities. Immunogold labeling of CB1 receptors isvisible on the membrane of axon terminals, labeled a1 and a2, filledwith electron-dense NPY immunoreactivity. N indicates the nucleus of aputative Schwann cell. Scale bars: 0.2 μm (scales for FIGS. 4B and 4Eare the same as in FIGS. 4A and 4D, respectively).

FIGS. 5A-B depict bar graphs showing the intrinsic effects of the CB1antagonist. SR141716A on capsaicin-evoked bronchospasm and cough.Results are expressed as mean±s.e.m, with n 6 for each condition.Asterisk indicates P<0.05.

FIG. 5A is a bar graph summarizing the bronchoconstricting effects ofcapsaicin (mg per animal, intratracheal) in the absence or presence ofthe CB1 antagonist SR141716A (0.2 mg per kg, i.v.).

FIG. 5B is a bar graph summarizing the tussigenic effects of capsaicin(0.3 μM, 4 min aerosol) in the absence or presence of SR141716A (0.2 mgper kg, i.v.).

FIGS. 6A-C depict the Ca²⁺-dependent biosynthesis of anandamide in ratlung tissue.

FIGS. 6A-B are representative high-performance liquidchromatography/mass spectrometry tracings for selected ionscharacteristic of endogenous anandamide (mass-to-charge ratio m/z=370,an adduct with Na⁺, [M+Na⁺]) and synthetic [2H₄]anandamide (m/z=374,[M+Na⁺]), which was added to the samples as an internal standard,respectively.

FIG. 6C is a bar graph summarizing the effects of EGTA (1 mM) or Ca²⁺ (3mM) on anandamide biosynthesis in rat lung membranes. Ca²⁺ significantlystimulated anandamide formation (mean±s.e.m.,*P<0.05, n=4).

FIGS. 7A-F depicts the structure and Ca²⁺ dependent biosynthesis ofanandamide precursors in rat lung tissue.

FIGS. 7A-B show the chemical structures ofalk-1-palmitoenyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-arachidonyl(NAPE 1) andalk-1-stearyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-arachidonyl(NAPE 2), respectively, two putative anandamide precursors.

FIGS. 7C-D are representative high-performance liquidchromatography/mass spectrometry tracings for selected ionscharacteristic of NAPE 1 (m/z=1009, deprotonated molecular ion, [M−H]⁻)and NAPE 2 (m/z=1039, [M−H]⁻), respectively.

FIGS. 7E-F are bar graphs summarizing that biosynthesis of NAPE 1 andNAPE 2, respectively, was significantly stimulated by incubation withCa²⁺ (3 mM) (mean±s.e.m.,*P<0.05, n=4) as compared to EGTA. NAPE 2 waseluted from the column as a doublet and the areas under both peaks werecombined for quantification.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides methods for ameliorating cough by means of (1)local administration of anandamide and other cannabinoid compounds, suchas those illustrated by formulae I, II, and III; and/or (2) local orsystemic administration of agents that increase the levels of endogenousor exogenously added cannabinoids in the upper respiratory tract byinhibiting cannabinoid inactivation, such as those represented byformulae IV and V.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The phrase “administration of a pharmaceutical composition” incorporatesthe phrases common usage and refers to any appropriate means to give apharmaceutical to a patient, taking into consideration the properties ofthe pharmaceutical composition and the preferred site of administration;e.g., in one embodiment, the pharmaceutical composition of the inventionis inhaled into the lungs.

The term “ameliorating” as used herein refers to any indicia of successin the treatment or amelioration of an injury, pathology, condition, orsymptom associated with a cough, including any objective or subjectiveparameter such as abatement; diminishing of symptoms or making thesymptom, injury, pathology or condition more tolerable to the patient;decreasing the frequency or duration of the symptom or condition;improving a patient's physical or mental well-being; and preventing theonset of the symptom or condition of coughing. The treatment oramelioration of symptoms can be based on any objective or subjectiveparameter; including, e.g., the results of a patient's observations,physical examination, or, simply an improvement in the patient's senseof well-being.

The term “anandamide” as used herein refers to arachidonylethanolamides(see, e.g., U.S. Pat. No. 5,631,297) an endogenous lipid that activatescannabinoid receptors and mimics the pharmacological effects ofΔ⁹-tetrahydrocannabinol, the active principle of hashish and marijuana.Anandamide analogs (equivalents) are described, e.g., in U.S. Pat. No.5,977,180; WO 99/60987; WO 99/64389. See also, e.g., U.S. Pat. Nos.6,028,084; 6,013,648; 5,990,170; 5,925,672; 5,747,524, 5,596,106; EP 0570 920, WO 94-12466.

The term “locally acting cannabinoid” as used herein refers tocannabinoids of the general formulae I, II, or III. These cannabinoidanalogs (equivalents) are described, e.g., in U.S. Pat. Nos. 5,635,530and 5,618,955.

The term “direct cannabinoid receptor agonist” as used herein refers toa compound that binds to and activates CB1-type cannabinoid receptors.

The terms “indirect cannabinoid receptor agonist,” “inhibitor ofcannabinoid inactivation,” or “cannabinoid inactivation inhibitor” asused herein refer to a compound that blocks the inactivating transportand/or degradation of cannabinoid compounds, consequently causing theaccumulation of the cannabinoid substances at its sites of action.

The term “endogenous cannabinoid” as used herein refers to an endogenousagonist, or an equivalent thereof, of a cannabinoid receptor.Cannabinoid receptors are described, e.g., in U.S. Pat. No. 6,013,648.Endogenous agonists include, e.g., 2-arachidonylglycerol or anandamide.See also U.S. Pat. Nos. 6,028,084; 6,017,919; 596,106; 5,990,170; and,Seltzman (1999) Curr. Med. Chem. 6:685-704.

The term “cough” as used herein refers to the act of coughing or to thepsychological or physiological sensations associated with causing acough that can be ameliorated by administration of a pharmaceuticalcomposition able to serve as a cannabinoid receptor agonist, to inhibitthe inactivating transport (e.g., the intracellular transport), and/orto inhibit anandamide hydrolysis of endogenous, or exogenously added,cannabinoid substances. The term “cough” is broadly defined and notlimited to a particular disease or cause creating this condition.

While the invention is not limited by any particular mechanism ofaction, in one embodiment, the invention contemplates administration ofthe locally acting cannabinoid receptor agonists and/or administrationof cannabinoid inactivation inhibitors, which inhibit the inactivatingtransport of cannabinoid substances, and/or inhibit the anandamidehydrolysis of cannabinoid substances, as described in further detail,below.

The phrase “inhibiting cannabinoid inactivation” means any measurableamount of increase in the amount of extracellular free cannabinoidsubstance. While the invention is not limited by any specific mechanism,the inhibition of inactivation can be accomplished by the pharmaceuticalcomposition by inhibition of inactivating uptake of the cannabinoidsubstance by the cell membrane or inhibition of anandamide hydrolysis.

The term “pharmaceutically acceptable excipient” incorporates the commonusage and includes any suitable pharmaceutical excipient, including,e.g., water, saline, phosphate buffered saline, Hank's solution,Ringer's solution, dextrose/saline, glucose, lactose, or sucrosesolutions, magnesium stearate, sodium stearate, glycerol monostearate,glycerol, propylene glycol, ethanol, and the like.

The terms “pharmaceutically effective” and “therapeutically effective”refer to a sufficient level of cough suppression or prevention in ahuman or animal resulting from the stimulation of cannabinoid orcannabinoid-like receptors. The term “therapeutically effective amount”and grammatical variations thereof refer to quantities of the activecompound that are sufficient to produce the desired therapeutic effectwhen delivered topically (e.g., by aerosol) or systemically (e.g.,orally).

General Methods

The methods of the invention use compounds capable of ameliorating coughby providing locally acting cannabinoids, inhibiting the inactivatingtransport of cannabinoids, and/or inhibiting anandamide hydrolysis. Avariety of exemplary compounds useful in these methods are describedherein. Exemplary routine methods for identifying these compounds aredescribed herein. Exemplary routine methods for identifying inhibitingthe inactivating transport of cannabinoids can be found in PatentApplication U.S. Ser. No 09/612,326, filed Jul. 6, 2000, the entirety ofwhich is incorporated herein by reference.

The skilled artisan will recognize that compounds useful in the methodsof the invention (e.g., arachidonylethanolamide) can be synthesizedusing a variety of procedures and methodologies, which are welldescribed in the scientific and patent literature., e.g., OrganicSyntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons,Inc., NY; Venuti (1989) Pharm Res. 6:867-873. The invention can bepracticed in conjunction with any method or protocol known in the art,which are well described in the scientific and patent literature.Therefore, only a few general techniques will be described prior todiscussing specific methodologies and examples relative to the methodsof the invention.

Structural Guidelines and Screening Tests to Design Locally ActingCannabinoids Useful in the Methods of the Invention

In the methods of the invention, pharmaceutical compositions comprisingcompounds capable of ameliorating cough are administered. Thesecompounds act by locally activating cannabinoid receptors. An exemplarylocally acting compound is an anandamide.

The biological actions of anandamide (arachidonyl-ethanolamide), anendogenous cannabinoid lipid, in cough and bronchospasm inhibition isthought to be through the anandamide's activation of the CB1 receptors.Such actions are theorized by studies described herein using CB1 and CB2receptor antagonists in the presence of locally acting cannabinoids. Theexperiments are described in greater detail below.

Furthermore, as described herein, the methods of the invention have beendemonstrated to ameliorate cough by using art-accepted animal models.Specifically, the cannabinoids and their anti-tussive effects have beenlooked at in guinea pigs, rats, and cats, (Gordon, R. et al. (1976) Eur.J. Pharmacol. Vol. 35 (2):309-313). In summary, these exemplarytechniques and guidelines provide clear parameters to select forcompounds useful as pharmaceutical compositions in the methods of theinvention.

Cannabinoids, such as anandamide, which can be found endogenously,produces a profound inhibition of cough and bronchial smooth musclecontraction, when administered either systemically or by localapplication in the airways. FIGS. 2A-C show bar graphs of the level ofbronchospasm and cough in guinea-pigs after administration of capsaicin,the pungent principle of chili pepper. Similar data were obtained inrats (intratracheal administration: 10 mg per animal, 41±6% of maximalbronchospasm; 30 mg per animal, 55±12%; 100 mg per animal, 81±19%;mean±s.e.m., n=3). This response was abrogated by anandamide when thecompound was administered systemically before capsaicin in guinea pigs(FIG. 2B). Similar results were obtained in rats (capsaicin, 10 mg peranimal, intratracheal administration: 37.2±4.2% of maximal bronchospasm;capsaicin after anandamide, 1 mg per kg, i.v., 14±7%; n=3). This effectwas completely reversed by the selective CB1 cannabinoid antagonistSR141716A, but only slightly reduced with a maximal dose of the CB2antagonist SR144528. Palmitylethanolamide, a structural analog ofanandamide that inhibits nociception in mice but does not interact withCB1 cannabinoid receptors as described in Calignano et al. (1998) Nature394, 277-281, was ineffective at alleviating capsaicin-evokedbronchospasm. When given as an aerosol to conscious guinea pigs,capsaicin stimulates C-fibre activity in the upper respiratory tract andtriggers cough. Systemic anandamide reduced capsaicin-evoked cough, aneffect that was abrogated by CB1 cannabinoid receptor blockade, as shownin FIG. 2C. Importantly, aerosolized anandamide also produced potentanti-tussive effects (FIG. 2C), which were not accompanied by any visualsigns of cannabinoid intoxication.

Anandamide had no direct bronchomotor action except at the highest dosetested (5 mg per kg), at which the compound elicited a smallbronchoconstriction (11.8±5.9% of maximal; mean±s.e.m., n=5). Toinvestigate further this response, the effects of anandamide inanesthetized rodents that were deprived of the bronchoconstricting toneconferred by the vagus nerve were examined. The effects are shown inFIGS. 3A-E. After vagotomy and administration of atropine (a cholinergicantagonist), which eliminate vagal influences, systemic application ofanandamide produced a dose-dependent bronchoconstriction in guinea pigsand rats (i.v.; 1 mg per kg, 0±0% of maximal bronchospasm; 3 mg per kg,12±1.7%; 5 mg per kg, 18.3±1.2%; n=3) in the presence and absence ofSR141716A, as shown in FIG. 3A. Similar effects were observed whenanandamide was injected into the guinea pig bronchi via a trachealcatheter (FIG. 3B), or applied to isolated strips of guinea pig lungparenchyma (FIG. 3C-E). FIG. 3C is a representative tracing illustratingthe effect of anandamide on muscle tension in guinea pig parenchymastrips and its reversal by the CB1 antagonist SR141716A. FIG. 3D is arepresentative tracing of the same lung strip of FIG. 3C responding tohistamine. FIG. 3E shows the contractions of lung parenchyma byanandamide and antagonism by SR141716A. The slow onset of the anandamideresponse in guinea pig lung strips is consistent with results obtainedin other isolated tissues, for example, as described in Devane, W. etal., (1992) Science 258, 1946-1949, while the low potency of anandamidein this preparation may be accounted for by limited tissue penetrationand/or rapid inactivation.

In agreement with this possibility, the anandamide transport inhibitorN-(4-hydroxyphenyl)-arachidonamide enhanced anandamide-evokedcontractions in guinea pig isolated lung strips (anandamide, 50 μM,0.336±0.07 dyne/mg of tissue; anandamide plusN-(4-hydroxyphenyl)-arachidonamide, 28 μM, 0.638±0.06 dyne/mg of tissue;P<0.05, n=6). The CB1 antagonist SR141716A blocked anandamidebronchoconstriction in vivo and in vitro (FIGS. 3A-E), whereas the CB2antagonist SR144528 had no such effect. The cannabinoid agonist HU210was also potent at eliciting guinea pig bronchial muscle constrictionafter tracheal administration (0.1 mg per animal, 10.0±0.6% of maximalbronchospasm; 1 mg per animal, 30±1.2%; 10 mg per animal, 60±2.2%; 30 mgper animal, 100%; n=6).

Anandamide has been claimed to activate vanilloid receptors. However,the vanilloid antagonist capsazepine had no effect on anandamide-evokedbronchospasm at a dose that completely prevented the capsaicin response(0.2 mg per kg, i.v.). These results indicate that removing the vagalexcitatory tone unmasked a bronchoconstricting activity of anandamidemediated through CB1 cannabinoid receptors.

The ability of anandamide to influence bronchial muscle contractilityafter local administration suggests that this compound may exert itseffects by activating CB1 cannabinoid receptors located within theairways. To test this possibility, the ultrastructural localization ofCB1 cannabinoid receptors in rat lungs by electron microscopy, wereexamined using an antibody directed against the intracellular C-terminusof the rat CB1 cannabinoid receptor protein. Immunogold stainingrevealed that cannabinoid receptors are present on nerve fibersdistributed amongst bronchial and bronchiolar smooth muscle cells, asshown in FIGS. 4A-C, or between the longitudinal and circular smoothmuscle layers, where several axons were packed together into glialcapsules (FIGS. 4D-E). All bundles contained at least one CB1cannabinoid receptor-positive axon.

Detailed evaluation (20 bundles consisting of 91 axons followed throughat least 25 consecutive sections) revealed that 36% of the axons werelabeled with the cannabinoid receptor antibody. The gold particleslabeling cannabinoid receptors were attached to the inner surface of theaxon plasma membrane, either at the release site or in the preterminalsegments. This is consistent with the fact that our antibody recognizesthe intracellular C-terminus of the CB1 cannabinoid receptor protein.Axon terminals bearing cannabinoid receptor immunoreactivity were inclose proximity to smooth muscle cells (0.2-0.5 mm) and contained alarge number of small agranular vesicles along with few dense-corevesicles (FIGS. 4A-B). In some cases, cannabinoid receptorimmunoreactivity was adjacent to clusters of vesicles accumulated at theplasma membrane, which most likely represent neurotransmitter releasesites (FIG. 4C).

In determining whether CB1 cannabinoid receptors are localized onnoradrenergic and/or non-noradrenergic fibers, a combination ofimmunogold staining for CB1 cannabinoid receptors and immunoperoxidasestaining for neuropeptide Y (NPY), a cotransmitter in sympatheticneurons, were used. We found that 63% of NPY-bearing axons were also CB1receptor-positive (FIGS. 4F-G). Importantly, however, extensive labelingwas observed on many NPY-negative axons, suggesting that bothnoradrenergic and/or non-noradrenergic nerves may express cannabinoidreceptors.

The finding that cannabinoid receptors are found predominantly, if notexclusively, on axon terminals of airway nerves, suggests thatanandamide regulates cough and bronchial smooth muscle tone through aprejunctional mechanism. Indeed, inhibition of excitatoryneurotransmission in the airways may provide a parsimonious explanationfor the ability of anandamide to oppose capsaicin-evoked cough andbronchospasm. This interpretation is further supported by the ability ofanandamide and other cannabinoid agonists to inhibit neurotransmitterrelease in peripheral tissues and in the central nervous system. Themechanism underlying the constricting actions of anandamide in animalslacking cholinergic control is currently unknown. One possibility, whichis consistent with the co-localization of CB1 cannabinoid receptors withNPY, is that anandamide inhibits the release of bronchodilatingmediator(s). Alternatively, anandamide may interact with cannabinoidreceptors on smooth muscle. The failure to detect CB1 cannabinoidreceptor immunoreactivity in lung smooth muscle may have been caused byinsufficient sensitivity of the technique used or by the presence insmooth muscle of a receptor variant that is not recognized by theantibody used. Interestingly, Northern blot analyses suggest thatalveolar type II cells in the lung may express two different CB1cannabinoid receptor mRNA species.

To test for the possibility that endogenous cannabinoids regulate airwayresponsiveness, the intrinsic effects of CB1 and CB2 antagonists onbronchospasm and cough in guinea pigs were determined. Blockade of CB1cannabinoid receptors with SR141716A had no bronchomotor consequencesper se, but significantly enhanced the bronchoconstriction and coughingevoked by capsaicin administered either via a tracheal catheter (FIGS.5A-B) or by i.v. injection (30 mg per kg; capsaicin alone, 55.3±8.2% ofmaximal bronchospasm; capsaicin after SR141716A [0.5 mg per kg, i.v.],92.3±3.4%; P<0.05, n=3). The CB2 antagonist SR144528 had no sucheffects. Although the bronchomotor actions of the CB1 antagonist may beaccounted for by its inverse agonist properties, two lines of evidencesuggest that this drug acted by opposing an ongoing cannabinoidmodulation. First, the lack of effect seen with the CB1 antagonist inthe absence of capsaicin is incompatible with an inverse agonistbehavior. Second, analyses by high-performance liquid chromatography(HPLC) coupled to positive-ionization electrospray mass spectrometry(MS) revealed that anandamide is synthesized in rat lung tissue througha Ca²⁺ ion-activated mechanism (FIGS. 6A-B). Rat lung membranes producedon average 0.6±0.2 pmol of anandamide per mg of protein in the presenceof the Ca²⁺ chelator, EGTA (1 mM); and 1.6±0.2 pmol of anandamide per mgof protein in the presence of Ca²⁺ (3 mM) (mean±s.e.m., n=4; P<0.05between EGTA and Ca²⁺; Student's t test) (FIG. 6C). Guinea pig lungmembranes produced 0.9±0.3 pmol of anandamide per mg of protein in thepresence of EGTA; and 8.8±1.2 pmol of anandamide per mg of protein inthe presence of Ca²⁺ (n 4; P<0.0001; Student's t test).

Anandamide is thought to originate from the enzymatic cleavage ofN-arachidonyl phosphatidylethanolamine (NAPE), the biosynthesis of whichis catalyzed by a Ca²⁺-dependent N-acyltransferase activity as describedin Di'Marzo, V. et al. (1994) Nature, 372, 686-691; Sugiura, T. et al.(1996) Eur. J. Biochem., 240, 53-62; Cadas, H. et al. (1997) J.Neurosci., 17, 1226-1242. Using negative ionization electrosprayHPLC/MS, two molecular species of NAPE in lipid extracts of rat lungmembranes:alk-1-palmitoenyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-arachidonyl(NAPE 1), depicted in FIG. 7A, andalk-1-stearyl-2-arachidonyl-sn-glycero-phosphoethanolamine-N-arachidonyl(NAPE 2), depicted in FIG. 7B were identified. Identifications werebased (1) on the occurrence of deprotonated molecules of appropriatemass (NAPE 1: mass-to-charge ratio (m/z) 1009; and NAPE 2, m/z 1039);and (2) on the chromatographic behavior of these components, which wassimilar to that of synthetic NAPE (FIGS. 7C-D). NAPE 1 and NAPE 2 weresynthesized by rat lung membranes in a Ca²⁺-dependent manner. Themembranes produced 1.5±0.02 pmol of NAPE 1 and undetectable levels inNAPE 2 when incubated with EGTA (1 mM); and 4.4±0.5 pmol of NAPE 1 and3.1±0.6 pmol of NAPE 2 when incubated with Ca²⁺ (3 mM) (n=4; P<0.05between EGTA and Ca²⁺) (FIGS. 7E-F). Guinea pig lung membranes alsoproduced NAPE 1 and NAPE 2 in a Ca²⁺-dependent manner. The presence inrodent lungs of a Ca²⁺-activated mechanism for the biosynthesis ofanandamide and its phospholipid precursor supports a role for thisendogenous cannabinoid in airway modulation. This suggests in turn thatinhibitors of anandamide inactivation (compounds of formulae IV and IV)may also produce cough inhibition and bronchodilation by means of theirability to cause accumulation of anandamide at its sites of action.

These results demonstrate that activation of CB1 cannabinoid receptorsby locally released cannabinoid compounds, such as anandamide,participates in the control of cough and bronchial contractility. Howcannabinoids exert their control on bronchial contractility may depend,however, on the state of the bronchial muscle. When the muscle iscontracted, as during capsaicin-evoked bronchospasm, anandamide maycounteract this contraction, possibly by inhibiting the prejunctionalrelease of excitatory neurotransmitters and neuropeptides. In contrast,when the smooth muscle is relaxed, as seen after removal of theconstricting influence of the vagus nerve, anandamides may causebronchoconstriction.

In summary, the present results suggest that local applications ofcannabinoid agents in the airways of animals whose vagal tone is notcompromised, result in cough inhibition. These effects are mediated byactivation of CB1 cannabinoid receptors located on peripheral terminalsof airway nerves. Furthermore, the results suggest that local orsystemic administration of inhibitors of endogenous cannabinoidinactivation may also result in cough inhibition. Since the animalmodels used in the present experiments are predictive of anti-tussiveactions/treatments in humans, it is anticipated that comparablecough-suppressing effects will be produced in human patients affected bypathological tussigenic conditions. These conditions include, but arenot limited to, persistent dry cough, cancer-induced cough, andangiotensin-converting enzyme (ACE) inhibitor-induced cough.

The present invention discloses a method for treating cough in a mammalthrough application of at least one direct or indirect cannabinoidreceptor agonist that is active at relieving or preventing cough by aperipheral action in the upper airways. The method comprises the step ofadministering to the mammal a cough-alleviating or cough-preventingamount of a pharmaceutical formulation comprising at least one compoundhaving the general formulae I, II, I, IV, or IV described below.

Formula I:

-   -   wherein    -   X is N—R1 or 0;    -   R is a saturated or unsaturated, chiral or achiral, cyclic or        acyclic, substituted or unsubstituted hydrocarbyl group with 11        to 29 carbon atoms, optionally incorporating up to 6 oxygen or        sulfur atoms;    -   R1, R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

When R2 is OH and X is N—H, they may be combined through the carbonylgroup to form a heterocyclic ring structure, e.g. an oxazolidinone ring.Alternatively, when R2 is OH and X is N—H, they may be combined to forma heterocyclic ring structure, e.g. a morpholine ring.

Formula II:

-   -   wherein    -   R is a saturated or unsaturated, substituted or unsubstituted        hydrocarbyl group with from 15 to 29 carbon atoms, optionally        incorporating up to 3 oxygen or sulfur atoms;    -   R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

When R2 is OH and X is N—H, they may be combined to form a heterocyclicring structure.

Formula III:

-   -   wherein    -   R is a saturated or unsaturated, substituted or unsubstituted        hydrocarbyl group with from 15 to 29 carbon atoms, optionally        incorporating up to 3 oxygen atoms;    -   R3 and R4 are selected independently from hydrogen, alkyl        (C1-4), alkenyl (C2-4), alkynyl (C2-4), cycloalkyl (C3-6), or        hydroxyalkyl group with from 2 to 4 carbon atoms;    -   R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4        carbon atoms; and    -   n is selected from 2 to 4.

Formula IV:

-   -   wherein    -   R is a polyunsaturated, substituted or unsubstituted hydrocarbyl        group with from 18 to 22 carbon atoms;    -   R2 is selected independently from substituted or unsubstituted        cycloalkyl (C3-6) group and substituted or unsubstituted phenyl        group (e.g., p-hydroxyphenyl, p-hydroxy-o-methyl-phenyl).        R₁—X—R₂  Formula V:    -   wherein    -   R1 is a saturated or polyunsaturated, substituted or        unsubstituted hydrocarbyl group with from 6 to 22 carbon atoms;    -   X is —C═O or SO₂—; and    -   R2 is a halogen or a halogen-substituted methyl group.        Pharmaceutical Formulations

The pharmaceutical compositions used in the methods of the invention canbe administered by any means known in the art, e.g., parenterally,topically, orally, or by local administration, such as aerosol ortransdermally. The pharmaceutical compositions can be formulated in anyway and can be administered in a variety of unit dosage forms dependingupon the condition, disease, degree, or cause of the cough, the generalmedical condition of the patient, the resulting preferred method ofadministration and the like. Routine means to determine drug regimens,formulations, and administration to practice the methods of theinvention are well described in the patent and scientific literature,see, e.g., the latest edition of Remington's Pharmaceutical Sciences,Maack Publishing Co, Easton Pa.

Pharmaceutical formulations can be prepared according to any methodknown to the art for the manufacture of pharmaceuticals. Thepharmacological composition of the invention can comprise other activeagents such as anti-inflammatory compounds. Pharmaceutically acceptablecompounds can contain a physiologically acceptable compound that acts tostabilize the composition or to increase or decrease the absorption ofthe agent and/or pharmaceutical composition. A formulation can beadmixtured with nontoxic pharmaceutically acceptable excipients that aresuitable for manufacture.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be obtained through combination ofcannabinoids of the invention with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable solid excipients such as carbohydrateor protein fillers include, e.g., sugars, including lactose, sucrose,mannitol, or sorbitol; starch from corn, wheat, rice, potato, or otherplants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, e.g., push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and a coatingsuch as glycerol or sorbitol. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g.,N-(4-hydroxyphenyl) arachidonamide) in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipientsinclude a suspending agent, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Oil-based pharmaceuticals are particularly useful for administration ofhydrophobic active agents. Oil-based suspensions can be formulated bysuspending an active agent (e.g., N-(4-hydroxyphenyl) arachidonamide) ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

The pharmaceutical formulations of the invention can also be in the formof oil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water can be formulated in admixture witha dispersing, suspending and/or wetting agent, and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those disclosed above. Additional excipients,e.g., sweetening, flavoring and coloring agents, can also be present.

In the methods of the invention, the pharmaceutical compounds can alsobe administered by intranasal or intrabronchial routes includinginsufflation, powders, and aerosol formulations (for examples of steroidinhalants, see Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa(1995) Ann. AllergyAsthma Immunol. 75:107-111). For example, aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They alsomay be formulated as pharmaceuticals for non-pressured preparations suchas in a nebulizer or an atomizer. Typically, such administration is inan aqueous pharmacologically acceptable buffer.

In the methods of the invention, the pharmaceutical compounds can bedelivered transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols.

In the methods of the invention, the pharmaceutical compounds can alsobe delivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of the drug,which is slowly released subcutaneously; see Rao (1995) J. Biomater Sci.Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations,see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheresfor oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674. Both transdermal and intradermal routes afford constantdelivery for weeks or months.

In the methods of the invention, the pharmaceutical compounds can beprovided as a salt and can be formed with many acids, including but notlimited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,succinic, etc. Salts tend to be more soluble in aqueous or otherprotonic solvents that are the corresponding free base forms. In othercases, the preferred preparation may be a lyophilized powder in 1 mM to50 nM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol at a pH range of4.5 to 5.5, that is combined with buffer prior to use.

In the methods of the invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration.These formulations will commonly comprise a solution of active agentdissolved in a pharmaceutically acceptable carrier. Acceptable vehiclesand solvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid canlikewise be used in the preparation of injectables. These solutions aresterile and generally free of undesirable matter. These formulations maybe sterilized by conventional, well-known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of active agent in theseformulations can vary widely and will be selected primarily based onfluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. For IV administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the invention can bedelivered by the use of liposomes which fuse with the cellular membraneor are endocytosed, e.g., by employing ligands attached to the liposome,or attached directly to the oligonucleotide, that bind to surfacemembrane protein receptors of the cell resulting in endocytosis. Byusing liposomes, particularly where the liposome surface carries ligandsspecific for target cells, or are otherwise preferentially directed to aspecific organ, one can focus the delivery of the active agent intotarget cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839;Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr.Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm.46:1576-1587.

In the methods of the invention, a pharmaceutical composition isadministered in an amount sufficient to ameliorate cough. The amount ofpharmaceutical composition adequate to accomplish this is defined as a“therapeutically effective dose.” The dosage schedule and amountseffective for this use, i.e., the “dosing regimen,” will depend upon avariety of factors, including the stage and/or severity of the disease,condition, or other cause of the cough, the severity of the cough, thegeneral state of the patient's health, the patient's physical status,age and the like. In calculating the dosage regimen for a patient, themode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra). The state of the art allows theclinician to determine the dosage regimen for each individual patient,active agent and disease or condition treated. Guidelines provided forsimilar compositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, e.g., dose schedule and dosage levels ofany CB1 cannabinoid receptor activator administered practicing themethods of the invention.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required and tolerated by thepatient. The formulations should provide a sufficient quantity of activeagent to effectively treat the cough. In one example, the concentrationof cannabinoid compounds, such as anandamide, in the pharmaceuticallyacceptable excipient is between about 0.1-100 mg per dose in an aqueoussolution. As another example, one typical pharmaceutical formulationsfor oral administration of N-(4-hydroxyphenyl) arachidonamide is in adaily amount of between about 0.5 to about 20 mg per kilogram of bodyweight per day. In an alternative embodiment, dosages are from about 1mg to about 4 mg per kg of body weight per patient per day are used.Lower dosages can be used, particularly when the drug is administered toan anatomically secluded site, such as the lung space, in contrast toadministration systemically into the blood stream. Substantially higherdosages can be used in topical administration. Actual methods forpreparing parenterally administrable formulations will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's, supra. See also Nieman, In “ReceptorMediated Antisteroid Action,” Agarwal, et al. (1987) eds., De Gruyter,New York.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

EXAMPLE 1

Chemical Synthesis

Anandamide and other fatty acid ethanolamides can be synthesizedfollowing standard procedures described in Giuffrida, A. et al. (1998)FEBS Lett., 422, 373-376. The synthesis of 2-arachidonyleglycerol andother monoacylglycerides can be accomplished as described in B.Serdarevich, (1967) Journal of the Oil Chemist Society, 44, 381-393. Thedrugs were dissolved in dimethylsulphoxide (DMSO), and administered inphysiological saline containing 10% DMSO.

EXAMPLE 2

Biological Assays

Bronchospasm. Dunkin-Hartley guinea pigs (Charles-River, weighing 200400g) or Wistar rats (Charles-River, weighing 200-300 g) were anesthetizedwith pentobarbital (40 mg per k.g., intraperitoneal) and fentanyl (25 mgper kg, intramuscular). The trachea and carotid artery were catheterizedto measure airway obstruction and systemic blood pressure. The jugularvein was catheterized to administer drugs. Pancuronium bromide (4 mg perkg, intravenous) was administered to prevent spontaneous breathing. Theanimals were ventilated with room air using a rodent ventilator (U.Basile, Comerio, Italy) run at 60 strokes per min; stroke volume was 3-7ml. Airway resistance was measured by using a differential pressuretransducer (U. Basile) connected by the side-arm of the trachealcatheter to a bronchospasm transducer. Bronchospasm was expressed as apercent of the maximal response, which was determined by clamping thetracheal catheter before and after each experiment. Drugs were dissolvedin saline containing 10% dimethylsulphoxide, and injected via thejugular vein. Responses were evaluated at their peak. Arterial bloodpressure was measured continuously with a pressure transducer connectedto a recorder (U. Basile). To abolish vagal influences on bronchialmusculature, in some experiments the vagus nerves were bilaterallytransected and administered with atropine sulfate (2 mg per kg, i.v.).

Cough. Conscious guinea pigs were individually exposed for 4 min toaerosolized capsaicin (0.3 mM) while recording coughs by using amicrophone placed in the exposure chamber as described in Bolser, D. C.,et al. (1995) (Eur. J. Pharmacol., 276, R1-R3. Each animal was treatedonly once with capsaicin. Aerosols were prepared with an Air ListerBasic apparatus (Hatù, Italy), regulated at an emission flow rate of 6liters per min.

Isolated lung strips. Guinea pig parenchimal strips were preparedessentially as described in Samhoun, M. N., et al. (1984)Prostaglandins, 27, 711-724. The strips were placed in 10-ml organ bathscontaining Krebs' buffer (in mM: NaCl, 118; KCl, 4; K2HPO4, 1.2; MgSO4,1.2; CaCl2, 2.5; NaHCO3, 25.0; glucose, 11.2; supplemented with 7 mMatropine sulfate and 15 mM indomethacin) at 37° C. and aerated with anoxygen/carbon dioxide mixture (95/5%). Muscle contractions were recordedwith an isometric force transducer (U. Basile) and expressed in dyne permg of fresh tissue.

EXAMPLE 3

Electron Microscopy The lungs were removed from 3 rats perfused with aphosphate-buffered (PB, 0.1 M) fixative containing 4% paraformaldehyde,0.2% picric acid and 0.05% glutaraldehyde, and were further fixed for 24h. Immunohistochemical analyses were conducted as described in Katona,I. et al. (1999) J. Neurosci., 19, 4544-4558. Rabbit C-terminal anti-CB1and anti-NPY antibodies were used at 1:5000 and 1:20,000 dilution,respectively. The specificity of the NPY antibody was reportedpreviously in Csiffary, A., et al. (1990) Brain Res. 506, 215-222.

EXAMPLE 4

Membrane Preparation and Lipid Extraction

Lung particulate fractions were prepared as described in Désarnaud, F.,et al. (1995) J. Biol. Chem., 270, 6030-6035. Incubations were performedfor 1 h at 37° C. in Tris buffer (50 mM, pH 7.4) containing CaCl₂ (3 mM)or EGTA (1 mM) and 2 mg per ml of membrane protein. Reactions werestopped by adding cold methanol and the lipids were extracted withchloroform. Before HPLC/MS analysis, anandamide and NAPE werefractionated by silica gel column chromatography as described in Cadas,H., et al. (1997) J. Neurosci., 17, 1226-1242.

EXAMPLE 5

High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS)

The anandamide was identified and quantified by reversed-phase HPLCcoupled to positive ionization electrospray MS, by using anisotope-dilution method described in Giuffrida, A., Rodriguez deFonseca, F., et al. (2000) Anal. Biochem., 280, 87-93. NAPE species werepurified by reversed-phase HPLC on a C18 Bondapak column (300×3.9 mmI.D., 5 mm) (Waters) maintained at 20° C. and interfaced with an AgilentHP1100 model mass spectrometer. HPLC conditions consisted of a lineargradient of methanol in water (from 75% to 100% methanol in 30 min) witha flow rate of 1 ml/min. Under these conditions, different NAPE specieswere eluted from the column as a group of peaks at retention timescomprised between 27 and 29 min. MS analyses were performed with theelectrospray ion source set in the negative ionization mode, the Vcapset at 5 kV, and the fragmentor voltage set at 200 V. Nitrogen was usedas a drying gas at a flow rate of 12 l/min. The drying gas temperaturewas set at 350° C. and the nebulizer pressure at 30 PSI. Forquantitative purposes, diagnostic ions (deprotonated molecular ions,(M−H)⁻) were extracted from full scan data and quantified by comparisonwith an external standard(1-palmityl-2-oleyl-sn-glycero-phosphoethanolamine-N-arachidonyl, AvantiPolar Lipids).

EXAMPLE 6

Data Analysis

Results are expressed as means±s.e.m. The significance of differencesamong groups was evaluated using Student's t test or analysis ofvariance followed by Dunnett's test.

All publications, Genebank references, patents, patent applicationscited herein are hereby expressly incorporated by reference for allpurposes. A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of ameliorating cough comprising the local administration tothe upper respiratory airways of a subject in need of such treatment ofa cannabinoid compound of formula I:

wherein X is N—R1 or 0; R is a saturated or unsaturated, chiral orachiral, cyclic or acyclic, substituted or unsubstituted hydrocarbylgroup, with 11 to 29 carbon atoms; R1, R3 and R4 are selectedindependently from hydrogen, alkyl (C1-4), alkenyl (C2-4), alkynyl(C2-4), cycloalkyl (C3-6), or hydroxyalkyl group with from 2 to 4 carbonatoms; R2 is OH or O—CO-alkyl, where the alkyl group has from 1 to 4carbon atoms; and n is selected from 2 to
 4. 2-34. (canceled)